The document summarizes TILLING (Targeting Induced Local Lesions IN Genomes), a technique that combines chemical mutagenesis with PCR to identify point mutations in genes of interest. It discusses how TILLING works, including generating a mutant population through chemical mutagen treatment, extracting DNA, pooling samples, amplifying target genes via PCR, detecting mutations via formation of heteroduplexes, enzymatically cleaving mismatches, and recovering phenotypes. Applications of TILLING include functional genomics, genetic engineering, and evaluating genetic diversity. Eco-TILLING is also summarized as a related technique for identifying natural genetic variation instead of induced mutations.

1. Assignment
On
Tilling & Eco-tilling
Submitted to
Dr. Baudh Bharti
Department of GPB, ANDUA&T, Kumarganj,
Ayodhya-224229
Submitted by
Mr. Prabhat Kumar Singh
Ph. D. Agril. Biotechnology 1st year
Course Title: Genomics in Plant Breeding
(GPB-605) 3(3+0)

2. CONTENTS
 What is TILLING
 Why TILLING
 Discovery of TILLING
 TILLING Procedure
 Application in plant species
 Merits and Demerits
 Eco-TILLING

3. TILLING
TARGETING INDUCED LOCAL LESIONS IN
GENOME

4. INTRODUCTION
 One of the most direct ways of establishing gene function is to identify a
mutation in the specific gene and to link this mutation to the phenotypic
change in the mutated organism.
 In the forward genetics approach (“from mutation through phenotype to
the gene”), large mutated populations have been created and screened for
alterations in the trait but this approach is both time-consuming and
labour-intensive.
 The reverse genetics strategy (“from gene sequence to phenotype”) has
widely replaced the forward approach in studies involved in detecting
gene function.

5. INTRODUCTION
 Several reverse genetics technologies, such as insertional mutagenesis
with T-DNA, transposon/retrotransposon tagging or gene silencing
using RNA interference, have been proposed for plant functional
genomics .
 However, the majority of these methods are fully applicable only for
model plants with small genomes, such as Arabidopsis or rice, and even
in these species, there are some drawbacks that limit their utilisation.

6. What is TILLING ?
TILLING is a general reverse genetic technique that combines chemical
mutagenesis with PCR based screening to identify point mutations in regions of
interest. (McCallum et.al, 2000)
TILLING is a powerful technology that employed heteroduplex analysis to detect
which organism in a population carry single nucleotide mutation in specific genes.
TILLING can also be used to detect naturally occurring SNP in genes among the
accession, variety or cultivar. To study the gene function, or to detect genetic marker
in population.
Reverse genetics is-
DNAsequence Protein Phenotypes
AGCTCAATCAGATAATC
TCGAGTTAGTCTATTAG

7. WHY TILLING ??
 Tool for functional genomics that can help decipher the functions
of the thousands of newly identified genes.
 To identify SNPs and/or INDELS in a gene of interest from
population.
 Genetic mutation is a powerful tool that establishes a direct
link between the biochemical function of a gene product and its
role in vivo.

8. Discovery of TILLING
 TILLING first began in the late 1990’s by McCallum who worked
on characterizing the function of 2-chromomethylase (CMT2)
gene in Arabidopsis.
 Claire McCallum utilized reverse genetic approaches such as T-
DNA lines and antisense RNA, but was unable to successfully
apply these approaches to characterize CMT2.

9. TILLING PROCEDURE
1. Generation of the mutant
population
2. Extraction of the DNA
3. Pooling
4. PCR orAmplification
5. Detection of heteroduplex
6. Heteroduplex Enzymatic
cleavage
7. Phenotypic Recovery

10. GENERATION OF MUTANT POPULATION
Mutagenizing seed with EMS done by soaking seeds in an
EMS solution (14-18 h in a 30-100 mM (0.3%-1%) EMS
solution.)
Planting the seeds in field
Mutagenized population (M1generation) is grown to maturity
allowed to self-fertilize to produce M2 seeds.
M2 seeds can be maintained as lines or bulked
If the M2 seed is bulked then lines need to be established using M3
seed. In either case, when sampling M2 plants to establishpopulation.

11. DNAEXTRACTION

12. POOLING

13. DNAAMPLIFICATION BY USING
PCR

14. DETECTION OF HETERODUPLEX
 Gene specific primers are available to find the sequence of gene of interest.
 Software are also use to identify the point mutation in a gene such as
CODDLE
 Codons Optimized to Detect Deleterious LEsions
 Once a genomic sequence and the corresponding coding sequence is available,
CODDLE identifies regions where point mutations are most likely to cause
deleterious effects on gene function.
 Screening the mutant the population compare with wild type at a single
nucleotide level.
 Detection of the cleavage fragment was based on a Denaturing High
Pressure Liquid Chromatography (DHPLC).

16. HETERODUPLEX ENZYMATIC
CLEAVAGE
 After the amplification of the desired sequence of heteroduplex, we
cleavage or cut at the point of mutation with the enzyme of S1 nuclease
familyCel1.

17. Cleaved products are detected on polyacrylamide denaturing
gels.

19. PHENOTYPE
RECOVERY
 When allelic series of mutation discovered
through TILLING
 Phenotypic analysis is required to verify the
effect of mutation.

20. Scheme depicting TILLING strategy applied to Arabidopsis

21. APPLICATIONS OF TILLING
Major areas of applications are
Functional genomics:
• The identification of numerous mutations in target region of genome.
Construction of TILLING library is useful for scientists to search for mutations
in gene of interest. TILLING offers a way to investigate target GOI in any crop
of interest without first having knowledge of gene product.
Genetic engineering:
• Agricultural interest in producing phenotypic variants without introducing
foreign DNA of any type into plants genome. T-DNA/ Transposon insertions are
used to obtain specific gene knockouts but practically limited to some crops
only. TILLING is in front of transgene, as consists of identification of numerous
mutations within a targeted region of whole genome.
Evaluation of genetic diversity of natural populations:
• Alternative to wild relatives, TILLING is used to introduce useful genetic
variation of elite germplasm. Also applicable in a population which has
several pre-existing polymorphism for developing SNPs.

22. Source: Muhammad Rashid et al, 2011

23. MERITS AND DEMERITS
OF TILLING
MERITS
• Cheap and rapid reverse
genetic approach
• Firstly use to detect a chemical
mutagenesis at single
nucleotide level
• It is independent of
genome size, reproductive
system or generation time.
• V
aluable for essential gene
DEMERITS
• Skilled labour is
required
• Depend upon primary
design
• Lower rate of
induction of mutation

24. • The first publication of the EcoTILLING method in whichTILLING
was modified to mine for natural polymorphisms was in 2004 from
work in Arabidopsis thaliana.
• EcoTILLING is similar to TILLING, except that its objective is to
identify natural genetic variation as opposed to induced mutations.
• Many species are not amenable to chemical mutagenesis; therefore,
EcoTILLING can aid in the discovery of natural variants and their
putative gene function
•This approach allows one to rapidly screen through many samples
with a gene of interest to identify naturally occurring SNPs and / or
small INs/DELS.
EcoTILLING

25. TILLING and EcoTILLING

27. • A study based on applying TILLING to elucidate gene function in a chemically
induced sorghum mutant population showed that total of five mutants were
detected for four gene targets.
• A total of 768 mutant lines were assayed for mutation induction in the target
genes.
• Four gene targets were selected based on their potential contribution to
bioenergy, nutrition and agronomic performance for high throughput
TILLING.
• Eight-fold pools of genomic DNA from leaf tissues of M2 plants were used for
TILLING.
• Mutagenesis sorghum inbred line BT*623 was used to generate the mutant
populations using EMS (0.1 to 0.6% mutagenesis).
• A subset of 768 mutant lines was analyzed by TILLING using four target
genes.
• A total of five mutations were identified resulting in a calculated mutation
density of 1/526 kb.

28. A gallery of selected mutant phenotypes